Abstract:During the past decade, several novel fluorescence microscopy techniques have emerged that achieve incredible spatial and temporal resolution beyond the diffraction limit. These microscopy techniques depend on altered optical setups, unique fluorescent probes, or post-imaging analysis. Many of these techniques also depend strictly on the use of unique fluorescent proteins (FPs) with special photoswitching properties. These photoswitchable FPs are capable of switching between two states in response to light. Al… Show more
“…In the years since the discovery, significant advances have been taken in discovering or developing new fluorescent protein photoswitches and mutants with characteristics tailored for specific experiments, including super-resolution fluorescence imaging applications. Several recent reviews summarize many of these developments in the context of super-resolution imaging [41][42][43][44][45].…”
Section: Fluorescent Proteins For Super-resolution Imagingmentioning
a b s t r a c tPhotoswitchable fluorescent probes are key elements of newly developed super-resolution fluorescence microscopy techniques that enable far-field interrogation of biological systems with a resolution of 50 nm or better. In contrast to most conventional fluorescence imaging techniques, the performance achievable by most super-resolution techniques is critically impacted by the photoswitching properties of the fluorophores. Here we review photoswitchable fluorophores for superresolution imaging with discussion of the fundamental principles involved, a focus on practical implementation with available tools, and an outlook on future directions.
“…In the years since the discovery, significant advances have been taken in discovering or developing new fluorescent protein photoswitches and mutants with characteristics tailored for specific experiments, including super-resolution fluorescence imaging applications. Several recent reviews summarize many of these developments in the context of super-resolution imaging [41][42][43][44][45].…”
Section: Fluorescent Proteins For Super-resolution Imagingmentioning
a b s t r a c tPhotoswitchable fluorescent probes are key elements of newly developed super-resolution fluorescence microscopy techniques that enable far-field interrogation of biological systems with a resolution of 50 nm or better. In contrast to most conventional fluorescence imaging techniques, the performance achievable by most super-resolution techniques is critically impacted by the photoswitching properties of the fluorophores. Here we review photoswitchable fluorophores for superresolution imaging with discussion of the fundamental principles involved, a focus on practical implementation with available tools, and an outlook on future directions.
“…The switching on/off of the fluorophore molecules in random or stochastic methods should be controlled so that a small subset of labelled fluorophores switches-on in each frame. This process can be controlled either by repeatedly switching the fluorophore on and off or by switching the fluorophore on and then bleaching it [53][54][55][56] . Using this approach, the achievable resolution in stochastically switching methods is down to 10-20 nm, far better than the diffraction limit of light (~200 nm).…”
Section: Nanoscopy With Wide-field Illumination (Palm Storm)mentioning
confidence: 99%
“…This improvement caused much excitement and made the single moleculebased nanoscopy methods popular among researchers that eventually led to the 2014 Nobel Prize in Chemistry. Many types of photoswitchable fluorophores are used in this method, these are reversibly or irreversibly switchable from one fluorescent state to another 2,56,57 . Both photochromic chemical dyes and genetically encoded fluorescent proteins (FPs) can be used, depending on the purpose.…”
Section: Nanoscopy With Wide-field Illumination (Palm Storm)mentioning
Optical nanoscopy has emerged as an important tool for live cell imaging at the nanoscale resolution in the field of life sciences. The 2014 Nobel Prize in Chemistry for this invention proves its importance in multidisciplinary areas of science. Several optical nanoscopic methods have been introduced in the past decade to achieve diffraction-unlimited resolution by implementing new optical setup or utilization of unique photoswitchable fluorophores, or both. In this review we extensively discuss the biological importance of nanoscopy and the latest advancements and types of fluorophores needed for imaging. This review will be a starter-kit for biologists working in the field of bioimaging.
“…PALM and STORM also require the ability to introduce photoswitchable dyes in specific target cells or sub-cellular units, which may be challenging in C. elegans. 65 Future research is likely to focus on the development of techniques that are feasible to implement in regular biology labs without special equipment and materials, and techniques that are scalable.…”
Microfluidics offers unique ways of handling and manipulating microorganisms, which has particularly benefited Caenorhabditis elegans research. Optics plays a major role in these microfluidic platforms, not only as a read-out for the biological systems of interest but also as a vehicle for applying perturbations to biological systems. Here, we describe different areas of research in C. elegans developmental biology and behavior neuroscience enabled by microfluidics combined with the optical components. In particular, we highlight the diversity of optical tools and methods in use and the strategies implemented in microfluidics to make the devices compatible with optical techniques. We also offer some thoughts on future challenges in adapting advancements in optics to microfluidic platforms.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.